This is the repository for the spatial (U01) hippocampus (HPC) project. The README.md contains a description of files in the repository including code and data to analyze the HPC data.

Study design

Experimental design to generate paired single-nucleus RNA-sequencing (snRNA-seq) and spatially-resolved transcriptomics (SRT) data in the human hippocampus. (A) Postmortem human tissue blocks from the anterior hippocampus were dissected from 10 adult neurotypical brain donors. Tissue blocks were scored and cryosectioned for snRNA-seq assays (red), and placement on Visium slides (Visium H&E, black; Visium Spatial Proteogenomics (SPG), yellow). (B) 10ξm tissue sections from all ten donors were placed onto 2-5 capture areas to include the extent of the HPC(n=36 total capture areas), for measurement with the 10x Genomics Visium H&E platform. (C) 10ξm tissue sections from two donors were placed onto 4 capture areas (n=8 total capture areas) for measurement with the 10x Genomics Visium-SPG platform. (D) Tissue sections (2-4 100ξm cryosections per assay) from all ten donors were collected from the same tissue blocks for measurement with the 10x Genomics 3’ gene expression platform . For each donor, we sorted on both and PI+NeuN+ (n=26 total snRNA-seq libraries).

Interactive Websites

All of these interactive websites are powered by open source software, namely:

We provide the following interactive websites, organized by dataset with software labeled by emojis:

Description of HPC data

This is a description of data files for this project.

  1. The MiSeq and NovaSeq folders has softlinks to the fastqs of slides V10B01−085 and V10B01−086
  2. The 2022-04-12_SPag033122 folder has softlinks to the fastqs of slides V11A20−297, V11L05−333, V11L05−335, V11L05−336, V11U08−081, V11U08−084.

Description of analyses of HPC data

REDCap

  • Script to extract HPC info only from the redcap form and extract all relevant (demographic/biological/rotation info etc) data to add to the spe object is here

01_spaceranger

Code to align reads using 10x SpaceRanger - Script to run space ranger with miseq and novaseq fastqs combined for samples V10B01−085 and V10B01−086 is here. - Script to run space ranger for all other samples is here.

02_build_spe

Code to build initial SPE, rotate/rearrange capture areas to better reflect anatomy, and drop spots - Script to build initial raw SpatialExperiment (SPE) object from spaceranger output is 01_raw_spe_allSamples.R. - Script to perform rotations and rearrange capture areas is 02_transform_spe_allSamples.R - Script to remove drop spots not in tissue section and spots with zero counts is 03_dropSpots.R.

03_shiny app_basic

Code to build temporary R Shiny app for initial manual annotations - Script to subset the basic_spe to make it memory effecient for shiny app is here. - Script to deploy the shiny app is here. - Scripts for running the shiny app is here.

04_QC

Code to perform QC on SRT data - Script to run QC is here - Script to plot results from QC is here

05_preprocess_batchCorrection

Code to preprocess data for clustering is here

06_clustering

Code to cluster SRT data. Primary method for clustering was PRECAST. Code for formatting data, running PRECAST, and visualizing results is here.

08_pseudobulk

Code to perform pseudobulk DE analysis is here

nnSVG

Code to identify spatially variable genes (SVGs) is here

spot_deconvo

Code for running spot-level deconvolution is here

NMF

Code to project NMF patterns learned in paired snRNA-seq data to Visium data and visualize results is here

enrichment_analysis

Code for running LDSC across spatial domains, snRNA-seq cell classes, and NMF patterns is here

Cell segmentation

VistoSeg here

cellpose here